Method of Forming Dendritic Cells from Embryonic Stem Cells

ABSTRACT

This invention relates to the culture of dendritic cells from human embryonic stem (ES) cells. Human ES cells are first cultured into hematopoietic cells by co-culture with stromal cells. The cells now differentiated into the hematopoietic lineage are then cultured with GM-CSF to create a culture of myeloid precursor cells. Culture of the myeloid precursor cells with the cytokines GM-CSF and IL-4 causes functional dendritic cells to be generated. The dendritic cells have a unique phenotype, as indicated by their combination of cell surface markers.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 13/364,074filed Feb. 1, 2012, which is a division of U.S. application Ser. No.12/876,830 filed Sep. 7, 2010 and issued as U.S. Pat. No. 8,133,732 onMar. 13, 2012, which is a division of U.S. application Ser. No.11/443,608 filed May 31, 2006 and issued as U.S. Pat. No. 7,811,821 onOct. 12, 2010 which claims the benefit of U.S. provisional application60/686,145, filed Jun. 1, 2005, which has since expired. All of theseapplications are incorporated by reference within.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under DAMD17-02-C-0130awarded by the DOD/DARPA. The government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

Embryonic stem cells are pluripotent cells capable of both proliferationin cell culture as well as differentiation towards a variety of lineagerestricted cell populations that exhibit multipotent properties(Odorico, et al., (2001) Stem Cells 19:193-204). Human embryonic stem(ES) cells are thus capable of commitment and differentiation to avariety of lineage-restricted paths resulting in very specific celltypes that perform unique functions.

Generally, ES cells are highly homogeneous, exhibit the capacity forself-renewal, and have the ability to differentiate into any functionalcell in the body. This self-renewal property can lead under appropriateconditions to a long-term proliferating capability with the potentialfor unlimited expansion in cell culture. Furthermore, it is understood,that if human ES cells are allowed to differentiate in an undirectedfashion, a heterogeneous population of cells is obtained expressingmarkers for a plurality of different tissue types (WO 01/51616;Shamblott, et al., (2001) Proc. Natl. Acad. Sci. U.S.A. 98:113). Thesefeatures make these cells a unique homogeneous starting population forthe production of cells having therapeutic utility.

There have been efforts by researchers in the field to develop methodsto culture a variety of progeny cell types from human ES cells. Forexample, U.S. Pat. No. 6,280,718 describes a method for culturing humanES cells into hematopoietic cells by culturing the human ES cell withstromal cells. Some methods of creating progeny cell types from human EScells involve the creation of embryoid bodies, which are threedimensional structures which can be formed by ES cells in culture andwhich foster the diverse differentiation of ES cells into variousdifferentiated progeny lineages. Other methods for creating progenylineages depend on the culturing of human ES cells with particularmedia, agents or types of cells to expose the ES cells to factors whichencourage differentiation in a particular direction. All these methodshave a common objective, which is to provide a source for particularcell types for scientific research and experimentation and, for somecell types, for ultimate transplantation into human bodies fortherapeutic purposes.

Dendritic cells are immune cells that perform a critical function in themammalian immune system. Dendritic cells (sometimes here DCs) arepowerful antigen-presenting cells which are present at low frequency intissues of the body in contact with the environment such as skin, andlinings of the nose, lungs, stomach and intestines. Dendritic cells havethe ability to uptake antigens and induce primary T cell responses toinitiate generalized immune system responses to pathogens. Dendriticcells are so named because of their long processes or arms, calleddendrites, that are characteristic of dendritic cell morphology.

Dendritic cells are generated continuously in the bone marrow from thehematopoietic lineage and mature in the blood. The dendritic cells of anindividual have heterogeneous phenotype and function. Dendritic cellsdevelop in several ways, and there may be differences among thedendritic cells depending on their lineage of derivation. Dendriticcells that develop from CD34+ hematopoietic progenitors along twoindependent pathways become Langerhans cells and interstitial dendriticcells. Dendritic cells derived from monocytes or from plasmocytoid Tcells are referred to as monocyte-derived DCs or plasmocytoid DCsrespectively. On the basis of their cellular origin phenotype, dendriticcells are normally classified broadly into two major divisions, myeloidor lymphoid. It was believed that myeloid DCs were developed from acommon myeloid precursor while lymphoid DCs developed from a commonlymphoid precursors, although it has now also been proposed that acommon myeloid DC precursor gives rise to all dendritic cell lineages.

The availability of human immature dendritic cells would be useful forthe study of antigen processing and presentation, as well as forunderstanding the mechanisms of the induction of immunity and tolerance.Functional analysis of human dendritic cell subsets was significantlyfacilitated by the development of in vitro systems for thedifferentiation of dendritic cells from CD34+ hematopoietic stem cellsand monocytes. However, using these existing protocols, obtaining largenumbers of human dendritic cell progenitors is a laborious process andis associated with potential risks for donors. Other aspects ofdendritic cell biology, such as dendritic cell ontogeny, have not beenstudied in humans due to the difficulties in obtaining tissues duringearly development. The advent of human ES cells represents anopportunity to overcome these limitations.

Functional dendritic cells have been generated from mouse ES cells usingembryoid bodies and by co-culture with mouse macrophagecolony-stimulating factor deficient bone-marrow stromal cell line, OP9.We have previously demonstrated that OP9 cells can be used to inducehematopoietic cells from human ES cells. The full potency of thosehematopoietic cells to produce progeny of the various lineages wasunexplored previously.

BRIEF SUMMARY OF THE INVENTION

In one embodiment, the present invention is a method of culturing humanembryonic stem cells into dendritic cells, the method comprising thesteps of co-culturing human embryonic stem cells with stromal cells thatdo not express macrophage colony-stimulating factor, wherein the stemcells are induced to differentiate into multipotent lympho-hematopoieticprogenitor cells and wherein the culture is not in the presence ofcytokines; culturing the progenitor cells with granulocyte/macrophagecolony stimulating factor (GM-CSF) to cause the expansion of myeloidprecursors cells; and recovering cells which have the phenotype ofimmature dendritic cells. Preferably the step of recovering cells withthe phenotype of dendritic cells includes culturing the myeloidprecursor cells with at least one cytokine selected from the groupconsisting of IL-4, TNF-α, IFN-α, and GM-CSF. Preferably, the stromalcells are OP9 cells and the culturing of step (b) is under non-adherentconditions.

In another embodiment, the present invention includes the step ofculturing the myeloid precursor cells with GM-CSF and TNFα or GM-CSF andIFN-α and recovering regulatory accessory cells, wherein the regulatoryaccessory cells are characterized by the markers CD1a^(low), CD9−,CD80^(low) and CD86 ^(low).

In another embodiment, the present invention includes the step ofculturing the myeloid precursor cells with GM-CSF and TNFα or GM-CSF andINFα and recovering regulatory accessory cells, wherein the regulatoryaccessory cells are characterized by the markers CD1a^(low), CD9,CD80^(low) and CD86 ^(low).

The present invention is also a culture of human dendritic cells, inwhich a majority of the cells in the culture have a phenotype of CD1a+,DC-SIGN+, CD4+, CD9^(low), CD11c+, CD40^(low), CD86+, CD80+, CD86+,HLA-ABC−, HLA-DR+, and are negative for CD207 and CD208. Preferably, atleast 70% of the cells in the culture have the phenotype.

In another embodiment, the invention is a culture of myeloid precursorcells in which a majority of the cells have a phenotype of myeloidprecursors and in which an excess of 90% of the cells are CD45+, CD4+,CD123^(low), negative for HLA-DR and include subpopulations of cellsexpressing MPO, M-CSFR, CD11b, CD11c, CD15 and CD16.

In another embodiment, the present invention is a method of making ofcellular vaccine, comprising differentiating human embryonic stem cellsinto population of dendritic cells, characterized by the markers CD1a,CD80, CD86, DC-SIGN, HLA-DR^(high), obtaining and preparing single cellsuspension of tumor cells from a patient, and fusing the embryonic stemcell-derived dendritic cells with the tumor cells so that a cellularvaccine is created.

Other embodiments of the present invention will be apparent to one ofskill in the art after review of the specification, claims and drawings.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIGS. 1A and B are schematic illustrations of the overall method of thepresent invention.

FIG. 2 illustrates the morphology and phenotypical features of myeloidprecursor cells generated in step 2 of FIG. 1. FIG. 2A is aphase-contrast micrograph of differentiated human ES cells growing inthe presence of GM-CSF. FIG. 2B is a Wright-stained cytospin of cellsobtained from that culture. FIG. 2C charts the colony forming cell (CFC)potential of the expanded cells (counts are mean of five experiments).FIG. 2D are graphs of data from representative experiments demonstratingexpression of surface and intracellular myeloid markers on GM-CSFexpanded human ES cells.

FIG. 3 illustrates the morphology and light scatter properties of hEScell-derived DSs. (A) Phase contrast micrograph of culture and (C)Wright-stained smears of differentiated H1 cells demonstrate numerousthin cytoplasmic processes (“veils”); (A) bar is 15 μm and (C) bar is 40μm. (B) When cultured on flat-bottom ultralow attachment plates, cellsform long dendrites; bar is 25 μm. (D) Light scatter properties andphenotype of cells obtained in step 3 after 9-day culture of hEScell-derived myeloid progenitors with GM-CSF and IL-4. Phenotypicanalysis from representative experiments using the H1 cell line showsthat R1-gated cells with a high scatter profile express CD1a and weaklyCD14.

DETAILED DESCRIPTION OF THE INVENTION

We report here that dendritic cells can be created in large numbers fromhuman ES cells. The co-culture system with a macrophagecolony-stimulating factor (M-CSF) deficient stromal cell line, such asthe murine line OP9, fosters the differentiation of human ES cells intohematopoietic cells. These hematopoietic cells have the capacity togenerate dendritic cells, a capacity which is exploited by GM-CSFculture of the hematopoietic cells. The dendritic cells derived fromhuman ES cells are morphologically, phenotypically and functionallycomparable to interstitial human dendritic cells naturally produced invivo.

Slukvin, et al., J. Immunology, 2006, 176:2924-2932, is an academicarticle by the inventors describing the present invention. It isincorporated by reference as if fully set forth below.

The overall method is schematically illustrated in FIGS. 1A and B, inwhich the process is broken down into three overall steps and isdemonstrated in its preferable form in the Examples below and inSlukvin, 2006, supra. By “multipotent lymphohematopoietic progenitorcells,” “myeloid dentritic cell (DC) precursor cells,” “immature DC,”“mature DC,” and “regulatory DC,” we mean the cell populations disclosedin FIG. 1B.

In Step 1, human ES cells are co-cultured with stromal cells, preferablyM-CSF deficient stromal cells, to induce differentiation of the cellsinto multipotent lymphohematopoietic progenitor cells. Preferably, thecells are OP9 cells.

In Step 2, the disassociated ES-derived cells from that culture are thencultured so that myeloid cell expansion occurs. Preferably, this is doneby culture of the cells with granulocyte/macrophage colony stimulatingfactor (GM-CSF) preferably as described below in the Examples. Alsopreferably, this step is performed in non-adherent conditions.Preferable non-adherent conditions require the tissue culture flask tobe coated with poly 2-hydroxyethyl methacrylate(HEMA, Sigma) asdescribed below. One could also prevent cell adherence by other means,such as cell shaking, using substances known to have non-adherentproperties to cover the plastic container or using commerciallyavailable non-adherent tissue flasks.

The result of this expansion step is a culture rich in myeloidprecursors, and that culture is then used in Step 3 to make dendriticcells by culture in serum free medium with GM-CSF and IL-4, or othercombination of cytokines (as described below) which conditiondevelopment to dendritic cells.

An optional separation procedure, which can be done with PERCOLLseparation, is shown between steps 2 and 3 and is used to remove bothclumps of cells and dead cells from the culture prior to inducingdendritic cell formation.

In the examples described below, dendritic cells are generated fromhuman ES cells by selective expansion of myeloid precursors obtained bythe co-culture of human ES cells with M-CSF-deficient stromal cellswithout cytokine addition. The hematopoietic cells resulting from theco-culture step are competent to be induced to differentiate intomyeloid precursor cells and then into immature dendritic cells.

A critical step in our protocol for generating DCs was the efficiency ofhematopoietic differentiation in human ES cell/OP9 co-culture.Co-cultures with a low number of CD34+CD43+Lin− multipotentlymphohematopoietic progenitors (less than 3%) failed to expand myeloidprecursors and, subsequently, differentiate to dendritic cells. “Lin−”indicates that these progenitors do not express CD11b, CD14, CD2, CD3,CD7, CD19, CD38, CD45RA, and HLA-DR markers present on more mature cellscommitted to specific hematopoietic lineage.

We used the entire cell suspension from the co-culture, rather thanisolated lymphohematopoietic precursors for the next step in whichgranulocyte/macrophage colony stimulating factor (GM-CSF) is used tomediate expansion of myeloid precursors which are capable ofdifferentiating into dendritic cells. In our hands, the most effectivefactor to cause the myeloid precursor cells to undergo this expansionwas GM-CSF, in contrast to other factors, such as SCF and FLT3-L, whichin our hands had little effect on the expansion of myeloid dendriticcell precursors.

The myeloid precursors derived from human ES cells and expanded withGM-CSF, contained myeloid colony-forming cells (CFCs), as well as smallpopulations of more mature cells with the dendritic cell phenotype.However, the majority of the cells morphologically resembled blasts ofthe myelomonocytic lineage and expressed CD4, CD45, CD123^(low) and lowlevels of CD 14. These cells were HLA-DR-negative. We found that thesecells included subpopulations of cells expressing MPO, M-CSFR, CD11b,CD11c, CD15, and CD 16.

The human ES cell-derived immature dendritic cells obtained by ourmethod had a phenotype of CD 1a+, DC-SIGN+, CD4+, CD9^(low), CD11c+,CD40^(low), CD80+, CD86+, HLA-ABC+, HLA-DR+, CD207− and CD208−, aphenotype comparable with interstitial dendritic cells differentiatedfrom the cord blood or bone marrow CD34+ hematopoietic stem cells.Preferably, at these immature dendritic cells comprise at least 70% ofthe cultured cells at this point. However, a distinct phenotypic featureof the human ES-derived dendritic cells was co-expression of CD14. Thelevel of CD14 expression was the lowest in cells differentiated usingIL-4, but was substantially higher on cells differentiated using TNF-α.Dendritic cells that develop from human CD34+ hematopoietic stem cellsin the presence of GM-CSF and TNF-α differentiate into Langerhans cellsand dermal and interstitial dendritic cells through intermediates thathave phenotypes that are CD1a+CD14− and CD1a-CD14+ respectively. So far,in our cultures, CD1a expression has always been associated with atleast low level expression of CD14, and we have not seen distinctCD1a+CD14− or CD1a-CD14+ populations in our cell cultures. Thus theculture conditions used for differentiation of the human ES cells intodendritic cells appears to use unique pathways that may not exactreplicates of the corresponding pathways of differentiation from CD45+hematopoietic stem cells in vivo.

The Examples below describe another embodiment of the present invention,a population of cells, wherein at least 70% are mature DCs.

Additionally, another embodiment of the invention is a population ofregulatory DC cells. Myeloid DC precursors cultured with GM-CSF andTNF-α or GM-CSF and IFN α develop into CD1a^(low), CD9−, CD80^(low),CD86^(low) accessory cells with low stimulatory activity. These cellscan represent regulatory DCs.

The co-culture system used here with the M-CSF deficient stromal cells(OP9 cells) differs from the system based on OP9 cells used with murineES cells. The method described here does not use a second co-culturewith the OP9 cells, unlike the mouse system. We collected the human EScell derivatives from the co-culture when the maximal amount of myeloidprogenitors were generated and then expanded those progenitors withGM-CSF in feeder-free non-adherent conditions. This technique resultedin the discrete population of dendritic cell precursors which is usefulfor further studies of dendritic cell development.

It has been shown recently that during embryoid body differentiation,cells expressing HLA-DR and capable of triggering proliferation of adultlymphocytes were generated. Zhan, et al., 2004, Lancet Jul 10;364(9429):163-71. However, the antigen-presenting properties and thephenotype of the cells generated in this system were not demonstrated.It is possible that cells obtained as described in this report weremacrophages. Our results provide, for the first time, evidence thathuman ES cells can be directly differentiated into cells withmorphology, phenotype and functional properties of antigen-presentingdendritic cells. Furthermore, this process is already relativelyefficient. We have been able to grow as many as 4×10⁷ dendritic cells ata time from 10⁷ initially plated human ES cells.

While dendritic cells have evident scientific interest and application,they also have potential use in human medicine. Several studies havedemonstrated that peptide-pulsed dendritic cells transferred in vivowere able to induce efficiently anti-tumor immune response in mice.These studies have encouraged subsequent development of dendriticcell-based vaccines for cancer immunotherapy in humans. In thesetechniques, immature dendritic cell precursors isolated from peripheralblood or dendritic cells generated from peripheral blood mononuclearcells and CD34+ hematopoietic progenitors are used in clinical trials ofdendritic cell based vaccines. However, these techniques are laborious,require repeated generation of new dendritic cells for each vaccinationand are difficult to standardize. Embryonic stem cells can be expandedwithout limit and can differentiate into multiple types of cells, andtherefore can be universal and scalable source of cells for dendriticcell vaccines. Potentially, dendritic cells with major HLA haplotypecombinations can be obtained from human ES cells to match donor MHChaplotype. In the clinical setting, human ES cell-derived dendriticcells would have several advantages over dendritic cells fromconventional sources. Large absolute numbers of dendritic cells could begenerated from the same donor cell line, and the same line of dendriticcells could be used for multiple vaccinations. Derivation of dendriticcells from human ES cells can be less laborious and more amendable forstandardization with implementation of bioreactor technology. Low riskof pathogen contamination and risk free donor collection are anotherimportant advantages of clinical use of human ES cell-derived dendriticcells.

In another embodiment, the present invention is a method of making acellular vaccine, comprising differentiating the human embryonic stemcells into a population of dendritic cells, characterized in that theyare CD1a⁺, CD80⁺, CD86⁺, DC-SIGN⁺, HLA-DR^(high), obtaining andpreparing single cell suspension of tumor cells from a patient, andfusing the embryonic stem cell-derived dendritic cells with cancercells. Gong, et al., J. Immunology, 2000, 165:1705-1711 and Parkhurst,et al., 2003, J. Immunology, 170:5317-5325 (both incorporated byreference) describe general techniques for cellular fusion.

In another embodiment, the invention is a method of forming a dendriticcell vaccine for treating of cancer, comprising dendritic cellsdifferentiating from human embryonic stem cells, where dendritic cellshave been fused with allogeneic cancer cells. One of skill in the artwould understand and appreciate the various methods of creating tumorvaccines. For example, U.S. Patent Application PublicationsUS2002/0131962 A1 and US2006/0063255 A1 disclose several methods.

In another embodiment, the present invention is a method of making adendritic cell vaccine for treating cancer, comprising differentiatinghuman embryonic stem cells into CD45+CD4+CD123^(low) myeloid precursorswhich include subpopulations of cells expressing CD11b, CD11c, and CD16,genetically altering the myeloid precursors to express immunogenic tumorproteins/peptides, and differentiating the genetically modified myeloidprecursors into immunogenic dendritic cells. For example, one may wishto transfect cells with tumor genes that will be the target of an immuneresponse. For example, one may wish to transfect cells withmelanoma-antigen-3 (MAGE-3), prostatic acid phosphatase (PAP) orprostate specific membrane antigen (PSMA).

In another embodiment, the present invention is a method of makingdendritic cells with tolerogenic properties which can be used fortreatment of rejection of human embryonic stem cell-derived tissuesobtained from the same cell line. By “tolerogenic properties,” we meanthat the cell suppresses rejection of a transplant by the host immunesystem. The cells will down-regulate a detrimental immune response ofthe host towards a transplanted tissue. For this purpose hEScell-derived myeloid precursors will be induced to differentiate intoregulatory DCs by culture with GM-CSF and TNF-α or GM-CSF and IFN α.

EXAMPLES Experimental Protocol and Results

Expansion of Human ES Cell-Derived Myeloid Progenitors with GM-CSF.

Recently we developed an in vitro culture system for hematopoieticdifferentiation from human ES cells, using cells of mouse M-CSFdeficient bone marrow stromal cell line OP9 as feeder cells, a step usedto start the protocols described here. Human ES cells were co-culturedwith OP9 cells so that they would differentiate into CD34+ cells whichare highly enriched in colony-forming cells and contain erythroid,myeloid, as well as lymphoid, progenitors and include a population ofCD34+ CD43+ Lin- multipotent hematopoietic progenitors. This step doesnot require cytokine addition. The maximal expansion of myeloidcolony-forming cells (CFCs) in the OP9 co-culture system was observed ondays 9 to 10 of differentiation. To induce selective expansion ofmyeloid progenitors, we harvested the resulting cells from days 9 or 10of human ES cell/OP9 co-culture and cultured the cells in non-adherentconditions in presence of GM-CSF. At the beginning of culture,aggregates of large cells were formed. Approximately 3 days afterinitiation of GM-CSF culture, individual cells appeared and rapidlyexpanded. After 9-10 days of culture with GM-CSF, and following theremoval of clumps and dead cells by PERCOLL separation, we obtained apopulation of cells of which 90% of the cells were CD45 positive. Morethan 90% of these CD45+cells contained intracellular MPO(myeloperoxidase, a marker of myeloid cells) but not TdT (terminaldeoxynucleotidyl transferase, a marker of lymphoid cells) and expresseda marker of myeloid progenitors, CD33. In addition, these human EScell-derived myeloid cells were CD4 positive, and weakly expressed IL-3receptor a-chain CD123. More than 50% of these cells expressed CD16,CD15, CD11b and CD11c (Table 2). Morphologically, the GM-CSF-expandedcells had multi-lobed or round nuclei and a moderate amount grayish,occasionally vacuolated, cytoplasm without visible granules (FIG. 2B),resembling bone marrow myelomonocytic precursors. Some of theGM-CSF-expanded cells retained myeloid CFC potential, but no erythroidor multi-lineage CFC potential was detected (FIG. 2C). In addition, arelatively small population of cells at advanced stages of maturationthat expressed a moderate level of CD14, low level of CD1a as well asthe HLA-DR, and CD80 and CD86 co-stimulator molecules were present(Table 2).

Cutaneous lymphocyte-associated antigen (CLA) expression on peripheralblood CD34+cells defines progenitors which further differentiate intoLangerhans' cells, while CD34+CLA- cell give rise to interstitialDC-like cells. No significant CLA expression was detected in the totalcell population obtained from OP9 co-cultures or isolated human EScell-derived CD34+ cells. However, CLA expression was found on a smallsubset of myeloid progenitors generated with GM-CSF.

GM-CSF appeared to be the most important factor in expansion of myeloidprecursors. Separately, the addition of SCF, FLT3L, or SCF with FLT3L toGM-CSF-supplemented cultures had little effect on total cell output andmyeloid CFCs numbers during 10 days of culture (Table 1). These datademonstrate that culture of differentiated human ES cells generated inOP9 system with GM-CSF predominantly expand into a unique population ofCD45+CD4+CD123^(low) myeloid precursors which include subpopulation ofcells expressing MPO, M-CSFR, CD11b, CD11c, CD15 and CD16.

Differentiation of Human ES-Cell Derived Myeloid Precursors intoDendritic Cells.

To induce differentiation of myeloid precursors into dendritic cells, wecultured the culture of precursor cells with GM-CSF and variouscombinations of IL-4, TNF-α, and IFN-α. In typical experiment, after7-10 days of culture with GM-CSF and IL-4, most of the cells appeared asclumps. In addition, individual floating cells with well-defineddendrites appeared in the cultures. Morphologically, these cells werelarge, had high nuclear cytoplasmic ratio, and had oval or kidney-shapednuclei and nonvacuolated, occasionally granular cytoplasm with very finecytoplasmic processes (FIG. 3A and C). Based on flow cytometric analysisof size and granularity, two cell populations were observed (FIG. 3D):R1, cells with high scatter profile and dendritic cell phenotype; andR2, cells with a low scatter profile, which lacked dendritic cellmarkers and which were more phenotypically similar to myeloidprogenitors generated in the second step. Dendritic cells identified asR1 gated cells expressed CD1a, DC-SIGN, CD4, CD11 c, HLA-ABC and HLA-DR,CD80, and CD86. Additionally, these cells expressed a low level of CD9,CD11b, CD123, and CD40. CD14 expression was very weak, but detectable,and most of the CD14-positive cells co-expressed CD1a. However all cellswere lacking CD83 expression.

In addition to IL-4, differentiation of myeloid precursors intodendritic cells was achieved by using other cytokines such as TNF-α andIFN-α or their combinations. However, most of the cells in cultures withTNF-α co-expressed low level of CD1a, high levels of CD14 and werelacking expression of CD9. In addition, in cultures with TNF-α, cellsdownregulated expression of costimulatory molecules. As expected,addition of IFN-α to these cell cultures resulted in increasedexpression of MHC class I molecules. However, IFN-a culture resulted ina decreased number of CD1a+cells, as well decreased CD14 expression.Similar to the monocyte-DC differentiation pathway, expression ofDC-SIGN on human ES cell-derived dendritic cells was primarily dependenton IL-4. Based on cell yield, phenotypic, and functional properties(Table 1 and 2), we concluded that a combination of GM-CSF and IL-4provides the best conditions for generation of functional dendriticcells from human ES cells.

By immunocytochemistry, human ES cell-derived dendritic cells werepositive for CD68, but not strongly so, and expressed a very low levelof intracytoplasmic but not membranous CD83. Fascin, an actin-bindingprotein that has been shown to be a highly selective marker of maturedendritic cells, was not detected. From this, we concluded that thedendritic cells generated by the process described so far were immature.To investigate whether these immature dendritic cells could be furthermatured, we treated cells generated from the above protocols withcalcium ionophore A23187. This treatment resulted in the up-regulationof CD83, CD86 and HLA-DR expression. The intensity of intracytoplasmicCD68 staining substantially increased and perinuclear condensation ofCD68 was evident in the cells so produced. In addition, some cellsbecame fascin-positive. LPS, TNF-α, IL-1β, PGE2, and IL-6 were notefficient in induction of maturation of hES cell-derived DCs. Takentogether, these data demonstrate that cells with typical dendritic cellmorphology and phenotype can be generated from human ES cells.

The dendritic cells induce allogeneic T Cell response and are capable ofantigen processing and presentation.

We next investigated to determine whether our human ES cell-deriveddendritic cells were fully functional as dendritic cells. As determinedby DQ ovalbumin assay, human ES cell-derived dendritic cells werecapable of taking up and processing antigen. Cells obtained in culturestreated with GM-CSF and IL-4 were the most efficient in antigenprocessing, while the dendritic cells differentiated with GM-CSF andTNF-α were less efficient.

A hallmark of the functionality of dendritic cells is their ability tostimulate naïve cells. By our tests, human ES cell-derived dendriticcells were able to trigger cord blood T cells, which are entirely naïve.Immature dendritic cells, generated in cultures with GM-CSF and IL-4added, were the most powerful stimulatory cells, while addition of TNF-αto the cell culture significantly diminished ability of the cells tostimulate naïve T lymphocytes. In addition, the dendritic cells wereable to stimulate adult donor T-cells.

To evaluate the capacity of dendritic cells to present antigens throughthe MHC class I pathway, we pulsed HLA-A02 H1 cell line-deriveddendritic cells with inactivated CMV virus and evaluated the ability ofthe cells to stimulate HLA-A0201 restricted CMV-specific T cell cloneHLA with specificity to CMV pp65 NLVPMVATV peptide. While the additionof dendritic cells to T-cells induced allogeneic response, a significantincrease in response by the T cells was obtained when cells werestimulated with CMV pulsed H1-derived dendritic cells (Table 3).Altogether, these data demonstrate that our culture system allowsgeneration of cells with phenotype, morphology and uniqueantigen-presenting properties characteristic of dendritic cells.

METHODS AND MATERIALS

Cell Lines, Cytokines and Monoclonal Antibodies (mAbs).

Human ES cell lines H1 (passages 32-51) and H9 (passages 40-44) weremaintained in an undifferentiated state by weekly passage on mouseembryonic fibroblasts. A mouse bone marrow stromal cell line OP9 wasobtained from Dr. Toni Nakano (Research Institute for MicrobialDiseases, Osaka University, Japan). This cell line was maintained ongelatinized 10 cm dishes (BD Bioscience, Bedford, Mass.) in OP9 growthmedium consisting of α-MEM (INVITROGEN, Carlsbad, Calif.), supplementedwith 20% defined fetal bovine serum (FBS; HyClone Laboratories, Logan,Utah). Sterile, recombinant, endotoxin and pyrogen-free SCF, FLT3-L,TNF-α, IL-4 were obtained from PeproTech (Rocky Hill, N.J.), GM-CSF fromBerlex Laboratories (Richmond, Calif.) and IFN-α from ScheringCorporation (Kenilworth, N.J.). The following mouse anti-human mAbswithout detectable cross-reactivity with murine cells have been used forflow cytometric analysis: CD1a-PE, CD4-PE, CD11b-FITC, CD16-PE,CD33-FITC, CD8O-PE, CD86-PE, HLA-DR-PE, myeloperoxidase (MPO)-FITC,terminal deoxinucleotidyl transferase (TdT)-FITC (Caltag, Burlingame,Calif.); CD9-PE, CD14-FITC, CD40-PE, CD43-FITC, CD45-PE, CD209(DC-SIGN)-FITC, CLA-FITC (BD Pharmingen); CD11c-PE, CD34-PerCP-Cy5.5(Becton Dickinson Immunocytometry Systems [BDIS], San Jose, Calif.);CD83-FITC, CD208 (DC-LAMP; Beckman Coulter, Miami, Fla.); CD123-FITC(Miltenyi Biotech, Auburn, Calif.); HLA-ABC-FITC (Sigma, St. Louis,Mo.); CD207 (Vector Laboratories).

Hematopoietic Differentiation of Human ES Cells in Co-Culture with OP9Cells.

The induction of human ES cells differentiation into hematopoietic cellswas done as previously described, Vodyanik, et al. 2005. Blood 105:617,which is incorporated herein by reference. Briefly, undifferentiatedhuman ES cells were harvested by treatment with 1 mg/ml collagenase IV(INVITROGEN) and added to OP9 cultures at approximate density of1.5×10⁶/20 ml per 10 cm dish in aMEM supplemented with 10% FBS (HyClone)and 100 μM Methyl β-D-thiogalactopyranoside (MTG) (Sigma, St. Louis,Mo.). Human ES cell/OP9 co-cultures were incubated for 9-10 days with ahalf medium change on days 4, 6, and 8 without added cytokines. Thehuman ES cells then differentiated into hematopoietic cells.

Generation of Human ES Cell-Derived Dendritic Cells.

A schematic diagram of the protocol used for generation of dendriticcells from human ES cells is depicted in FIG. 1. On day 9-10 of human EScell/OP9 co-culture, differentiated derivatives of human ES cells wereharvested by treatment with collagenase IV (INVITROGEN; 1 mg/ml inα-MEM) for 20 min at 37° C., followed by treatment with 0.05%Trypsin-0.5 mM EDTA (INVITROGEN) for 15 min at 37° C. After trypsininactivation by FBS, these cells were re-suspended in α-MEM supplementedwith 10% FBS (HyClone) and 100 ng/ml GM-CSF, and transferred into tissueculture flasks (BD Bioscience) coated with poly 2-hydroxyethylmethacrylate (HEMA, Sigma) to prevent cell adherence. The cells werethen cultured for 8-10 days with a half medium change every fourth dayto expand dendritic cell precursors. To evaluate the effect of SCF andFLT3-L on the expansion of these human ES cell-derived dendritic cellprecursors, we cultured the cells in the presence of (1) 100 ng/mlGM-CSF +20 ng/ml SCF; (2) 100 ng/ml GM-CSF+50 ng/ml FLT3-L; or (3) 100ng/ml GM-CSF+20 ng/ml SCF+50 ng/ml FLT3-L. Subsequently, the cells werespun over 20% PERCOLL (Sigma) to remove dead cells and cell aggregates.As a third step, PERCOLL-isolated cells were cultured for 7-9 days inHEMA-coated flasks in StemSpan® serum-free expansion medium (SFEM; StemCell Technologies, Vancouver, Canada) supplemented with lipid mixture 1(Sigma) and 100 ng/ml GM-CSF, with the addition of the followingcytokines: (1) 100 ng/ml IL-4, (2) 20 ng/ml TNF-α, (3) 10⁴ U/ml IFN-α,and (4) 100 ng/ml IL−4+20 ng/ml TNF-α Cells were cultured for 7-9 dayswith a half medium change every fourth day. To further maturatedendritic cells, we cultured the cells obtained in step 3 in SFEM mediumwith 400 ng/ml of A23187 calcium ionophore (Sigma) for 48 hours.

Flow Cytometry Analysis

Cells were prepared in PBS-FBS (PBS containing 0.05% sodium azide, 1 mMEDTA, and 2% FBS), supplemented with 2% normal mouse serum (Sigma), andlabeled with a combination of mAbs. Samples were analyzed using aFACSCalibur™ flow cytometer (BDIS) with CellQuest™ acquisition software(BDIS). List mode files were analyzed by FlowJo software (Tree Star,Inc., Ashland, Oreg.). Control staining with appropriate isotype-matchedcontrol mAbs (BD Pharmingen) was included to establish thresholds forpositive staining and background linear scaled mean fluorescenceintensity (MFI) values. The percentage (%) of positive cells wascalculated as % of positive cells stained with specific mAb-% ofbackground staining with corresponding isotype control. ΔMFI wascalculated as MFI of cells stained with specific mAb-MFI of cellsstained with corresponding isotype control. Linear scaled MFI was usedas an indicator of relative antigen density on given cells.

Antigen Processing Assay

Ovalbumin (OA) processing assays were performed using self-quenchedconjugate of ovalbumin (DQ-OVA; Molecular Probes, Eugene, Oreg.) thatexhibits bright green fluorescence upon proteolytic degradation.Dendritic cells obtained as in step 3 of FIG. 1 were incubated with 100μg/ml DQ-OVA for 30 min at 37° C. in DMEM/F12 (INVITROGEN) containing 2%FBS, and 1% of non-essential amino acids. Cells incubated at 4° C. wereused as a control for background fluorescence. OVA proteolysis wasevaluated by flow cytometry.

Clonogenic Progenitor Cell Assay

Hematopoietic clonogenic assays were performed in 35 mm low adherentplastic dishes (StemCell Technologies) using a 1 ml/dish of MethoCult™GF with H4435 semisolid medium (StemCell Technologies) consisting of 1%methylcellulose, 30% FBS, 1% BSA, 50 ng/ml SCF, 20 ng/mlgranulocyte-macrophage colony stimulating factor (GM-CSF), 20 ng/mlIL-3, 20 ng/ml IL-6, 20 ng/ml granulocyte colony stimulating factor(G-CSF), and 3 units/ml erythropoietin. All clonogenic progenitor assayswere performed in duplicates and CFCs were scored after 14-21 days ofincubation according to their colony morphology as erythroid (E-CFC),granulocyte, macrophage, megakaryocyte (GEMM-CFC),granulocyte-macrophage (GM-CFC), granulocyte (G-CFC) and macrophage(M-CFC). The frequency of CFC was calculated per 10⁶ total cells.

Allogenic Mixed Lymphocyte Reaction (MLR)

Adult mononuclear cells were isolated from peripheral blood samplesobtained from healthy laboratory volunteers by density gradientcentrifugation on Histopaque-1077. Mononuclear cord blood cells werealso purchased from Cambrex Bio Science (Walkersville, Md.). Themononuclear cells were depleted of monocytes by plastic adherence andused as responder cells. Graded numbers (1×10³ to 3×10⁴/well) ofirradiated (35 Gy) stimulatory cells were co-cultured with 1x10⁵responder cells for 6 days in 96-well flat bottom plates (Corning) inRPPMI 1640 containing 5% human AB serum (Sigma). [3H]thymidine (Sigma)was added (1 μCi/well) during the last 16 hours of incubation. Cellswere harvested onto glass fiber filters and incorporation of[3H]thymidine was measured by scintillation counting.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is understood that certain adaptations of theinvention are a matter of routine optimization for those skilled in theart, and can be implemented without departing from the spirit of theinvention, or the scope of the appended claims.

TABLE 1 Relative cell yield after each culture step* Relative Cell YieldStep 1 8.8 ± 4.4 Step 2 GM-CSF 5.5 ± 3.7 GM-CSF + SCF 5.6 ± 5.7 GM-CSF +FLT3-L 4.6 ± 4.2 GM-CSF + SCF + FLT3-L 5.1 ± 5.0 Step 3 GM-CSF + IL-43.3 ± 4.1 GM-CSF + TNF-α 2.3 ± 1.7 GM-CSF + IFN-α 2.3 ± 1.6 GM-CSF +IL-4 + TNF-α 1.9 ± 1.3 *Relative cell yield at each step calculated as anumber of cells obtained from one initially plated undifferentiatedhuman ES cell (total number human ES cells plated on OP9/total number ofcells obtained after corresponding step); results calculated as mean ±SD of 4 to 10 experiments.

TABLE 2 Phenotypic analysis of DCs induced by different cytokinecombinations* Step 3 Step 3 Step 3 Step 3 Cell GM-CSF + GM-CSF +GM-CSF + GM-CSF + subset Step 2 IL-4 TNF-α IL-4 + TNF-α IFN-α R1 gated %NA 58.8 ± 12.3 45.5 ± 12.1 46.7 ± 14.9 39.9 ± 7.5  cells CD1a %  3.3 ±2.1 82.9 ± 12.4 66.9 ± 24.0 78.2 ± 7.7  30.3 ± 27.1 ΔMFI 750.2 ± 700.774.8 ± 60.8 148.3 ± 161.9 77.1 ± 72.1 CD14 % 12.6 ± 7.1 25.6 ± 7.5  71.1± 12.2 39.0 ± 19.3 19.8 ± 15.1 ΔMFI 14.7 ± 4.2 27.6 ± 15.5 55.3 ± 38.131.5 ± 29.0 60.7 ± 50.8 DC- % <1 87.6 ± 7.7  <2 84.7 ± 4.2  17.3 ± 15.4SIGN ΔMFI 460.3 ± 352.0 213.8 ± 160.1 40.2 ± 39.1 CD83 % <1 <1 <1 <1 <1CD11c %  60.0 ± 14.2 94.1 ± 5.3  98.0 ± 1.6  93.7 ± 3.3  91.0 ± 8.5 ΔMFI 132.1 ± 59.9 282.3 ± 37.2  202.3 ± 19.8  237.6 ± 17.8  97.4 ± 41.8CD11b %  59.4 ± 13.1 67.4 ± 29.0 48.8 ± 24.9 56.0 ± 5.4  59.6 ± 8.4 ΔMFI  69.3 ± 23.0 52.9 ± 33.6 24.6 ± 14.3 47.9 ± 32.5 40.1 ± 35.3 CD123%  35.5 ± 14.6 58.8 ± 12.3 63.5 ± 16.6 45.1 ± 7.9  29.4 ± 18.6 ΔMFI 27.8 ± 15.2 35.9 ± 14.6 28.3 ± 12.6 33.9 ± 20.1 18.9 ± 15.3 HLA- % 79.6± 8.8 90.3 ± 8.4  91.8 ± 4.1  84.8 ± 9.3  99.2 ± 0.9  ABC ΔMFI 125.7 ±61.6 92.4 ± 10.6 130.3 ± 62.1  111.2 ± 55.4  258.0 ± 47.9  HLA-DR % 14.9 ± 12.0 90.1 ± 6.3  90.1 ± 4.1  82.1 ± 8.0  89.4 ± 7.7  ΔMFI 189.5± 83.7 597.0 ± 204.3 267.3 ± 123.1 208.3 ± 82.9  509.8 ± 340.2 CD86 %35.1 ± 9.1 93.4 ± 3.5  85.4 ± 7.3  90.1 ± 2.9  82.4 ± 14.1 ΔMFI  60.2 ±24.3 1767.4 ± 1122.3 158.5 ± 94.6    439 ± 131.0 125.3 ± 107.2 CD80 % 7.9 ± 7.8 81.2 ± 21.8 84.8 ± 10.7 81.8 ± 11.6 81.6 ± 19.3 ΔMFI 621.2 ±492.9 128.9 ± 80.4  295.8 ± 353.7 61.0 ± 13.2 CD40 %  4.6 ± 4.4 46.4 ±16.9 43.3 ± 23.7 57.0 ± 1.6  53.9 ± 26.8 ΔMFI 27.0 ± 11.4 16.6 ± 5.2 47.2 ± 32.5 21.0 ± 10.2 Cell Step 2 Step 3 Step 3 Step 3 Step 3 subsetGM-CSF + GM-CSF + GM-CSF + GM-CSF + IL-4 TNF-α IL-4 + TNF-α IFN-α*Results are mean ± SD of 4 to 5 independent experiments; for step 3cultures % and ΔMFI of R1 gated cells calculated.

TABLE 3 Antigen-presenting capacity of H1-derived DCs* T Cells DC CMVProliferation (cpm) IFN-γ (pg/ml) + − −  652 ± 129 0 + + − 17225 ± 579 224 ± 26.7 + + + 20303 ± 1279 326 ± 11.8 *HLA-A02 H1-derived dendriticcells (cells obtained in step 3 with GM-CSF + IL-4) incubated overnightwith or without CMV virus and then added to the HLA-A0201 restrictedallogeneic T cell clone with specificity to CMV pp65. Results expressedas a mean ± SD of triplicate.

We claim:
 1. A method of culturing human embryonic stem cells intodendritic cells, the method comprising the steps of: (a) co-culturinghuman embryonic stem cells with stromal cells that do not expressmacrophage colony-stimulating factor, wherein the stem cells are inducedto differentiate into multipotent lympho-hematopoietic progenitor cellsand wherein the culture is not in the presence of cytokines; (b)culturing the progenitor cells with granulocyte/macrophage colonystimulating factor (GM-CSF) to cause the expansion of myeloid precursorscells; and (c) recovering cells which have the phenotype of immaturedendritic cells.
 2. The method of claim 1 wherein the step of recoveringcells with the phenotype of dendritic cells includes culturing themyeloid precursor cells with at least one cytokine.
 3. The method ofclaim 2 wherein the cytokine is selected from the group consisting ofIL-4, TNF-α, IFN-α, and GM-CSF.
 4. The method of claim 1 wherein thestromal cells are OP9 cells.
 5. The method of claim 1 wherein theculturing of step (b) is under non-adherent conditions.
 6. The method ofclaim 1 further including, between step (b) and step (c), the step ofseparating clumps of cells and dead cells from the cell culture.
 7. Themethod of claim 1, additionally comprising the step of culturing themyeloid precursor cells with GM-CSF and TNFα or GM-CSF and INFα andrecovering regulatory accessory cells, wherein the regulatory accessorycells are characterized by the markers CD1a^(low), CD9, CD80^(low) andCD86^(low).
 8. The method of claim 1 wherein the expansion of themyeloid precursor cells is under serum-free conditions.
 9. A culture ofhuman dendritic cells, in which a majority of the cells in the culturehave a phenotype of CD1a+, DC-SIGN+, CD4+, CD9^(low), CD11c+,CD40^(low), CD86+, CD80+, CD86+, HLA-ABC+, HLA-DR+, and are negative forCD207 and CD208.
 10. A culture of myeloid precursor cells in which amajority of the cells have a phenotype of myeloid precursors and inwhich at least 90% of the cells are CD45+, CD4+, CD123^(low), negativefor HLA-DR and include subpopulations of cells expressing MPO, M-CSFR,CD11b, CD11c, CD15 and CD16.
 11. A culture of human regulatory DCs inwhich majority of the cells express CD1a^(low), CD80^(low), CD86^(low)phenotype and display diminished ability to induce the proliferation ofnaïve T cells.
 12. A method of making of cellular vaccine, comprising:a. differentiating human embryonic stem cells into a population ofdendritic cells, characterized by the markers CD1a, CD80, CD86, DC-SIGN,HLA-DR^(high); b. obtaining and preparing single cell suspension oftumor cells from a patient; and c. fusing the embryonic stemcell-derived dendritic cells with the tumor cells so that a cellularvaccine is created.
 13. A method of making a dendritic cell vaccine fortreating cancer, comprising: a. differentiating human embryonic stemcells into CD45+CD4+CD123^(low) myeloid precursors which includesubpopulations of CD11+, CD11c+, CD16+ cells; b. altering the myeloidprecursor cells to express immunogenic tumor proteins or peptides; andc. differentiating the genetically modified myeloid precursors intoimmunogenic dendritic cells.
 14. The method of claim 13 wherein thealtered myeloid precursor cells express a peptide selected from thegroup consisting of MAGE3, PAP and PSMA.
 15. A method of makingdendritic cells with tolerogenic properties which can be used fortreatment of rejection of human embryonic stem cell-derived tissuesobtained from the same cell line, comprising the steps of: a.differentiating hES cells into myeloid precursor cells; and b. obtainingregulatory dendritic cells, wherein the cells are characterized byCD1a^(low), CD9−, CD80^(low), CD86^(low), by culturing the myeloidprecursors with GM-CSF and TNF α or GM-CSF and IFNα.